Over the past several years, we have developed a unique ensemble of resources by which to explore fundamental issues in the earliest stage of intestinal neoplasia, the formation and maintenance of the adenoma. Our studies in the Min mouse strain have highlighted two new pathways by which the activity of the tumor suppressor gene Apc is lost: homologous somatic recombination and apparent epigenetic silencing. These early pathways do not involve the canonical genetic instability mechanisms involving mismatch repair or[unreadable] chromosome loss. We are now positioned to search for polymorphisms in the mouse and human that affect somatic recombination or epigenetic silencing. These studies have also highlighted the preponderance of polyclonality in the early intestinal adenoma. We now aim to investigate whether this polyclonality extends to tumors that involve epigenetic silencing and[unreadable] desmoid fibromas associated with defects in the Apc and p53 tumor suppressor genes. Finally, our studies of the familial intestinal adenoma in the mouse have identified two molecular modifiers of[unreadable] net tumor growth rate - DNA cytosine methylase and a secretory phospholipase. Though each modifier has only modest activity when introduced singly, in combination they show very strong synergy. We shall investigate the molecular basis of such synergy and explore how broadly it is found. We have positioned ourselves[unreadable] to pursue the more global identification of molecular modifiers through mutagenesis of the mouse germline and the use of an isogenic strategy that avoids the complications of abundant polymorphic modifiers. Intestinal neoplasia becomes life-threatening when it progresses to invasive and metastatic forms. We have[unreadable] established conditions in which the tumors of Min mice are locally invasive and methods for MicroCT imaging that permit longitudinal studies of progression and regression. Over the coming years, we aim to deepen our studies of early and late intestinal neoplasms by seeking molecular markers expressed within tumors and in the serum of tumor-bearing animals. The former goal will be explored not only by array analysis, but also by retroviral tagging. The latter goal will be explored by the emergent power of mass spectrometry.[unreadable] [unreadable] AIMS[unreadable] Aim la: Does a total deletion of Apc show the classical Min phenotype in heterozygotes, Apcde'/+?[unreadable] Aim 1 b: In ApcMin/+ (Mid+) heterozygotes do any adenomas form that retain an active, functional wildtype Apc allele?[unreadable] Aim Ic Does desmoid fibroma formation in Mid+ heterozygotes also involve loss of the wildtype Apc allele?[unreadable] Aim Id: Does the silencing pathway involve methylation of the promoter controlling expression of the wildtype Apc allele?[unreadable] Aim le: Is silencing limited to the wildtype allele, or does it also affect the Min mutant allele of Apc?[unreadable] Aim If: Is there a heterozygous effect of the Min nonsense allele that[unreadable] predisposes to adenoma formation?[unreadable] [unreadable] Aim 2a: Is early adenoma formation in the Min/+ intestinal epithelium polyclonal (cf Novelli et al., 1996)? What is the clonality of the contrasting Min-induced desmoid fibroma when localized and when invasive?[unreadable] Aim 2b: Are rates of mitotic segregation affected by the Apc genotype or by the genetic background?[unreadable] Aim 2c: Does transforming growth factor alpha (TGFa) affect the multiplicity, phenotype and clonality of Min-induced adenomas?[unreadable] Aim 2d: In the Min mouse, does homologous somatic recombination play a major role in loss-of-heterozygosity, as opposed to chromosomal instability?[unreadable] Aim 2e: Are the allele losses that are seen in human colonic adenomas also a result of homologous somatic recombination?[unreadable] [unreadable] Aim 3a: Is the secretory phospholipase (PlaZgZa; see MacPhee et al., 1995, and Gould et al., 199613) a component of the modifier-of-Min, Mom?? Do the components of Mom7 (Mom7A and Mom?@ correspond to members of the cluster of phospholipases in this region of the genome?[unreadable] Aim 3b: While working toward the analysis of Mom? at the molecular level, to establish which allele of Mom7A and of Mom7B is functional, resistance or sensitivity?[unreadable] Aim 3c: Is the Min phenotype modified in animals homozygous for a targeted allele that inactivates Wnt2 and/or in animals heterozygous for a P-catenin mutation?[unreadable] Aim 3d: Do any of the known molecular modifiers of the Min phenotype Morn? affect the proliferative and apoptotic dynamics of the stem cell and proliferative region of the normal intestinal epithelium or within the nascent adenoma?[unreadable] Aim 3e: Does the action of any of these modifiers depend upon the Paneth cell compartment?[unreadable] [unreadable] Aim 4a: Does the Mom? effect on the adenoma pathway depend upon wildtype p53 function? Does Mom1 affect desmoid tumor formation or growth?[unreadable] Aim 4b: Does DNA cytosine methyltransferase (Dnmt) deficiency affect the desmoid pathway?[unreadable] Aim 4c: Do Morn? and Dnmt act independently on the adenoma pathway?[unreadable] [unreadable] Aim 5a: To validate and characterize further a set of candidate dominant Enhancers (5) and Suppressors (7) of the Min survival phenotype found in our initial sensitized two-generation screen for mutagen-induced modifier alleles.[unreadable] Aim 5b: To develop the screen in a compact form to facilitate the isolation and mapping of mutagen-induced dominant Enhancers and Suppressors lying in vital genes.[unreadable] Aim 5c: For a DNA sequence whose methylation status decreases in Min induced adenomas, test whether targeted inactivation of the gene enhances the Min phenotype. retinoblastoma cell cycle regulator Rb influences the Min phenotype.[unreadable] Aim 5d: Investigate whether heterozygosity for a knockout allele of the retinoblastoma cell cycle regulator Rb influences the Min phenotype.[unreadable] Aim 5e: Analyze two new polymorphic modifier loci linked to the Apc locus on mouse chromosome 18 - a recessive enhancing allele in the strain BTBR, and a semidominant resistance allele in the strain DBN2.[unreadable]